Abscisic acid (ABA) is a plant hormone that regulates signal transduction associated with abiotic stress responses (Cutler et al., 2010, Abscisic Acid: Emergence of a Core Signaling Network. Annual Review of Plant Biology 61:651-679). ABA regulates numerous physiological processes and plays a major role in abiotic stress responses and tolerance to water deficit (i.e., drought). ABA biosynthesis is stimulated by decreases in soil water content, which lead to elevated hormone levels that in turn stimulate large-scale alterations in transcript abundance, guard cell closure, increased production of protective osmolytes, and numerous other physiological changes (Cutler et al., 2010).
A land-plant specific signaling pathway composed of receptors, phosphatases and kinases mediates ABA responses (Cutler et al., 2010). ABA elicits many of its cellular responses by binding to a soluble family of receptors called PYR/PYL proteins. PYR/PYL proteins belong to a large family of ligand-binding proteins named the START superfamily (Iyer et al., 2001; Ponting et al., 1999). These proteins contain a conserved three-dimensional architecture consisting of seven anti-parallel beta sheets that surround a central alpha helix to form a “helix-grip” motif. Together, these structural elements form a ligand-binding pocket for binding ABA or other agonists.
In the ABA response pathway, the phosphorylation status of three closely related ABA-regulated SnRK2 protein kinases is tied to environmental stress. When activated by phosphorylation on a critical activation loop near their ATP-binding site, these kinases phosphorylate downstream transcription factors, ion channels and most likely other proteins involved in ABA action (Weiner et al., 2010). Under ideal growth conditions the SnRK2s are continuously dephosphorylated and inactivated by a family of protein phosphatases (clade A PP2Cs), which results in nearly undetectable SnRK2 kinase activity in the absence of abiotic stress. When PP2C activity is inhibited by ABA-bound receptors, SnRK2s become highly active, probably because of their intrinsic ability to autoactivate by cis- and trans-autophosphorylation on their activation loops (Ng et al., 2011). Thus, ABA ultimately controls SnRK2 activity by receptor-mediated inhibition of PP2C activity.
The ABA signaling pathway has been exploited to improve plant stress response and associated yield traits via numerous approaches (Yang et al., 2010). The direct application of ABA to plants improves their water use efficiency (Raedmacher et al., 1987). Natural ABA can be used for improving drought tolerance in horticultural species (U.S. Pat. Publ. No. 2008/0227645) as well as several other uses. ABA analogs with improved resistance to metabolic degradation have been disclosed (U.S. Pat. Publ. No. 2008/0200339; U.S. Pat. No. 6,004,905; U.S. patent application Ser. No. 14/385,695), and these compounds have provided more persistent effects than ABA itself.
Because of their potential for improving crop yield (Notman et al., 2009), the discovery of ABA agonists has received increasing attention (Park et al., 2009; Melcher et al., 2010, Identification and mechanism of ABA receptor antagonism. Nature Structural & Molecular Biology 17(9):1102-1110). The first synthetic ABA agonist identified was the naphthalene sulfonamide pyrabactin (Park et al., 2009), which efficiently activates ABA signaling in seeds but has limited activity in vegetative tissues, where the most critical aspects of abiotic stress tolerance occur. Sulfonamides highly similar to pyrabactin have been disclosed as ABA agonists (see U.S. Pat. Publ. No. 2013/0045952) and abiotic stress modulating compounds (see U.S. Pat. Publ. No. 2011/0230350). Non-sulfonamide ABA agonists have also been described (see U.S. Pat. Publ. Nos. 2013/0045952 and 2011/0271408).
In Arabidopsis, the sulfonamide agonist quinabactin preferentially activates the three dimeric ABA receptors PYR1, PYL1 and PYL2. Its activity on these receptors is sufficient to induce drought tolerance, guard-cell closure, and ABA-mediated gene expression. Moreover, it has activity in crop species including soybean and maize. The activity of quinabactin in Arabidopsis is abolished in a mutant pyr1; pyl1;pyl2,pyl4 strain that removes the three ABA receptors with which quinabactin has highest potency. The preferential activity of quinabactin on the dimeric receptors combined with the necessity of these receptors for quinabactin action suggests that activating the dimeric ABA receptors is sufficient to elicit the major effects associated with ABA action.
Although quinabactin points to the dimeric receptors as targets for chemical control of ABA action and drought tolerance, genetic data indicate that the monomeric ABA receptors also play roles in ABA signaling. For example, a pyl8 loss-of-function mutants strain shows reduced root-growth inhibition by ABA (Antoni et al., 2012). In addition, the sequential removal of monomeric ABA receptors from a strain lacking the three dimeric receptors increases ABA insensitivity in proportion to the number of monomeric receptors removed (Gonzalez-Guzman et al., 2012). This argues that the ABA receptors may play additive roles in ABA signaling; however, the relative importance of each receptor class (dimeric versus monomeric) to ABA signaling is not clear, since mutant strains that selectively remove multiple monomeric receptors have not yet been described. Nonetheless, the monomeric receptors make contributions to ABA sensitivity and, as such, compounds that activate monomeric receptors may be useful for controlling drought tolerance, either on their own or in combination with compounds that activate the dimeric receptors such as quinabactin.
The present invention provides an N-acylsulfonamide scaffold that affords access to potent agonists of diverse monomeric ABA receptors. Compounds that preferentially activate PYL5 or PYL9 are disclosed as well as compounds that activate multiple monomeric receptors with relatively lower selectivity. Members of this compound class are bioactive in vivo; they are capable of inhibiting seed germination and can close guard cells in planta, a key physiological response necessary for chemical control of drought tolerance. The ability of this compound class to induce ABA responses in a plant demonstrates that monomeric receptors can be targeted for chemical control of ABA responses.